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- PDB-6ud0: Solution-state NMR structural ensemble of human Tsg101 UEV in com... -

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Basic information

Entry
Database: PDB / ID: 6ud0
TitleSolution-state NMR structural ensemble of human Tsg101 UEV in complex with K63-linked diubiquitin
Components
  • (Ubiquitin) x 2
  • Tumor susceptibility gene 101 protein
KeywordsCELL CYCLE / ESCRT-I / viral budding / ubiquitin E2 variant
Function / homology
Function and homology information


positive regulation of viral budding via host ESCRT complex / positive regulation of ubiquitin-dependent endocytosis / extracellular transport / ESCRT I complex / regulation of extracellular exosome assembly / negative regulation of epidermal growth factor-activated receptor activity / viral budding / regulation of MAP kinase activity / exosomal secretion / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway ...positive regulation of viral budding via host ESCRT complex / positive regulation of ubiquitin-dependent endocytosis / extracellular transport / ESCRT I complex / regulation of extracellular exosome assembly / negative regulation of epidermal growth factor-activated receptor activity / viral budding / regulation of MAP kinase activity / exosomal secretion / protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / positive regulation of exosomal secretion / membrane fission / ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway / multivesicular body assembly / Flemming body / virion binding / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I / positive regulation of protein monoubiquitination / mitochondrion transport along microtubule / fat pad development / endosome to lysosome transport / female gonad development / seminiferous tubule development / negative regulation of epidermal growth factor receptor signaling pathway / viral budding via host ESCRT complex / male meiosis I / viral release from host cell / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / autophagosome maturation / energy homeostasis / keratinocyte differentiation / regulation of neuron apoptotic process / regulation of proteasomal protein catabolic process / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / Constitutive Signaling by NOTCH1 HD Domain Mutants / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / multivesicular body / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / Regulation of FZD by ubiquitination / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / APC-Cdc20 mediated degradation of Nek2A / Regulation of innate immune responses to cytosolic DNA / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / NF-kB is activated and signals survival / Downregulation of ERBB2:ERBB3 signaling / Pexophagy / NRIF signals cell death from the nucleus / VLDLR internalisation and degradation / Regulation of PTEN localization / Activated NOTCH1 Transmits Signal to the Nucleus / neuron projection morphogenesis / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Regulation of BACH1 activity / TICAM1, RIP1-mediated IKK complex recruitment / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Translesion synthesis by REV1 / Translesion synthesis by POLK / regulation of mitochondrial membrane potential / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / InlB-mediated entry of Listeria monocytogenes into host cell / Regulation of activated PAK-2p34 by proteasome mediated degradation / Josephin domain DUBs / HCMV Late Events / Translesion synthesis by POLI / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / IKK complex recruitment mediated by RIP1 / Gap-filling DNA repair synthesis and ligation in GG-NER / ubiquitin binding / PINK1-PRKN Mediated Mitophagy / positive regulation of protein ubiquitination / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / TNFR1-induced NF-kappa-B signaling pathway / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / N-glycan trimming in the ER and Calnexin/Calreticulin cycle
Similarity search - Function
Steadiness box (SB) domain / Vps23 core domain / Steadiness box (SB) domain profile. / Ubiquitin E2 variant, N-terminal / : / UEV domain / UEV domain profile. / ESCRT assembly domain / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like ...Steadiness box (SB) domain / Vps23 core domain / Steadiness box (SB) domain profile. / Ubiquitin E2 variant, N-terminal / : / UEV domain / UEV domain profile. / ESCRT assembly domain / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Phosphatidylinositol 3-kinase Catalytic Subunit; Chain A, domain 1 / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin-like (UB roll) / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily / Roll / Alpha Beta
Similarity search - Domain/homology
Polyubiquitin-B / Tumor susceptibility gene 101 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsStrickland, M. / Watanabe, S. / Bonn, S.M. / Camara, C.M. / Fushman, D. / Carter, C.A. / Tjandra, N.
CitationJournal: Structure / Year: 2021
Title: Tsg101/ESCRT-I Recruitment Regulated by the Dual Binding Modes of K63-Linked Diubiquitin
Authors: Strickland, M. / Watanabe, S. / Bonn, S.M. / Camara, C.M. / Fushman, D. / Carter, C.A. / Tjandra, N.
History
DepositionSep 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 22, 2021Group: Database references / Category: citation / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Jun 14, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.status_code_nmr_data
Revision 1.3Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ubiquitin
B: Ubiquitin
C: Tumor susceptibility gene 101 protein


Theoretical massNumber of molelcules
Total (without water)33,9303
Polymers33,9303
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: NMR spectroscopy was used to determine the quaternary structure.
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1target function

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Components

#1: Protein Ubiquitin


Mass: 8604.845 Da / Num. of mol.: 1 / Mutation: K63R
Source method: isolated from a genetically manipulated source
Details: Distal domain of K63-linked diubiquitin. The K63R mutation is made to prevent formation of polyubiquitin during enzymatic linkage to the proximal domain. Joined to the proximal domain of ...Details: Distal domain of K63-linked diubiquitin. The K63R mutation is made to prevent formation of polyubiquitin during enzymatic linkage to the proximal domain. Joined to the proximal domain of diubiquitin at residue G76 using an isopeptide bond.
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0CG47
#2: Protein Ubiquitin


Mass: 8691.918 Da / Num. of mol.: 1 / Mutation: +D77
Source method: isolated from a genetically manipulated source
Details: Proximal domain of K63-linked diubiquitin. The additional aspartic acid on the C-terminus (+D77) is made to prevent formation of polyubiquitin during enzymatic linkage to the distal domain. ...Details: Proximal domain of K63-linked diubiquitin. The additional aspartic acid on the C-terminus (+D77) is made to prevent formation of polyubiquitin during enzymatic linkage to the distal domain. Joined to the distal domain of diubiquitin at residue K63 using an isopeptide bond.
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0CG47
#3: Protein Tumor susceptibility gene 101 protein / ESCRT-I complex subunit TSG101


Mass: 16633.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSG101 / Plasmid: pET-28B / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 / References: UniProt: Q99816
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic22D 1H-15N HSQC
188isotropic22D 1H-15N HSQC
122isotropic12D 1H-15N HSQC
199isotropic12D 1H-15N HSQC
133isotropic12D 1H-15N HSQC
11010isotropic12D 1H-15N HSQC
144isotropic12D 1H-15N HSQC
155anisotropic12D 1H-15N HSQC
166isotropic12D 1H-15N HSQC
177anisotropic12D 1H-15N HSQC

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1200 uM [U-98% 15N] Tsg101 UEV domain, 200 uM K63-linked diubiquitin, distal domain, 200 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of Tsg101 UEV domain (bound form)15NTsg_CSPs_bound93% H2O/7% D2O
solution8200 uM [U-98% 15N] Tsg101 UEV domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of Tsg101 UEV domain (free form)15NTsg_CSPs_free93% H2O/7% D2O
solution2200 uM Tsg101 UEV domain, 200 uM [U-98% 15N] K63-linked diubiquitin, distal domain, 200 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-distal domain of diubiquitin (bound form)15NDistal_CSPs_bound93% H2O/7% D2O
solution9200 uM [U-98% 15N] K63-linked diubiquitin, distal domain, 200 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-distal domain of diubiquitin (free form)15NDistal_CSPs_free93% H2O/7% D2O
solution3200 uM Tsg101 UEV domain, 200 uM K63-linked diubiquitin, distal domain, 200 uM [U-98% 15N] K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-proximal domain of diubiquitin (bound form)15NProx_CSPs_bound93% H2O/7% D2O
solution10200 uM K63-linked diubiquitin, distal domain, 200 uM [U-98% 15N] K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OSample used for measuring chemical shift perturbations of 15N-proximal domain of diubiquitin (free form)15N_Prox_CSPs_free93% H2O/7% D2O
solution4125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe distal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Lu-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (reference).15NTsg_PCS_Dist_Lu93% H2O/7% D2O
solution5125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe distal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Tm-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (paramagnetic).15NTsg_PCS_Dist_Tm93% H2O/7% D2O
solution6125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe proximal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Lu-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (reference).15NTsg_PCS_Prox_Lu93% H2O/7% D2O
solution7125 uM [U-98% 15N] Tsg101 UEV domain, 125 uM K63-linked diubiquitin, distal domain, 125 uM K63-linked diubiquitin, proximal domain, 20 mM potassium phosphate, 50 mM sodium chloride, 93% H2O/7% D2OThe proximal domain of diubiquitin was mutated (T14C) to incorporate a covalently bound Tm-M8-DOTA-SPy tag for the measurement of pseudocontact shifts in Tsg101 UEV domain (paramagnetic).15NTsg_PCS_Prox_Tm93% H2O/7% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
200 uMTsg101 UEV domain[U-98% 15N]1
200 uMK63-linked diubiquitin, distal domainnatural abundance1
200 uMK63-linked diubiquitin, proximal domainnatural abundance1
20 mMpotassium phosphatenatural abundance1
50 mMsodium chloridenatural abundance1
200 uMTsg101 UEV domain[U-98% 15N]8
20 mMpotassium phosphatenatural abundance8
50 mMsodium chloridenatural abundance8
200 uMTsg101 UEV domainnatural abundance2
200 uMK63-linked diubiquitin, distal domain[U-98% 15N]2
200 uMK63-linked diubiquitin, proximal domainnatural abundance2
20 mMpotassium phosphatenatural abundance2
50 mMsodium chloridenatural abundance2
200 uMK63-linked diubiquitin, distal domain[U-98% 15N]9
200 uMK63-linked diubiquitin, proximal domainnatural abundance9
20 mMpotassium phosphatenatural abundance9
50 mMsodium chloridenatural abundance9
200 uMTsg101 UEV domainnatural abundance3
200 uMK63-linked diubiquitin, distal domainnatural abundance3
200 uMK63-linked diubiquitin, proximal domain[U-98% 15N]3
20 mMpotassium phosphatenatural abundance3
50 mMsodium chloridenatural abundance3
200 uMK63-linked diubiquitin, distal domainnatural abundance10
200 uMK63-linked diubiquitin, proximal domain[U-98% 15N]10
20 mMpotassium phosphatenatural abundance10
50 mMsodium chloridenatural abundance10
125 uMTsg101 UEV domain[U-98% 15N]4
125 uMK63-linked diubiquitin, distal domainnatural abundance4
125 uMK63-linked diubiquitin, proximal domainnatural abundance4
20 mMpotassium phosphatenatural abundance4
50 mMsodium chloridenatural abundance4
125 uMTsg101 UEV domain[U-98% 15N]5
125 uMK63-linked diubiquitin, distal domainnatural abundance5
125 uMK63-linked diubiquitin, proximal domainnatural abundance5
20 mMpotassium phosphatenatural abundance5
50 mMsodium chloridenatural abundance5
125 uMTsg101 UEV domain[U-98% 15N]6
125 uMK63-linked diubiquitin, distal domainnatural abundance6
125 uMK63-linked diubiquitin, proximal domainnatural abundance6
20 mMpotassium phosphatenatural abundance6
50 mMsodium chloridenatural abundance6
125 uMTsg101 UEV domain[U-98% 15N]7
125 uMK63-linked diubiquitin, distal domainnatural abundance7
125 uMK63-linked diubiquitin, proximal domainnatural abundance7
20 mMpotassium phosphatenatural abundance7
50 mMsodium chloridenatural abundance7
Sample conditionsIonic strength: 0.1 M / Label: Conditions_1 / pH: 5.8 / Pressure: 1 atm / Temperature: 300 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCEBrukerAVANCE6001Cryoprobe
Bruker AVANCEBrukerAVANCE8002Cryoprobe

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Processing

NMR software
NameVersionDeveloperClassification
NMRPipe9.5Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
TopSpin3Bruker Biospincollection
CcpNmr Analysis2.4.2CCPNpeak picking
CcpNmr Analysis2.4.2CCPNchemical shift assignment
X-PLOR NIH2.51Schwieters, Kuszewski, Tjandra and Clorerefinement
X-PLOR NIH2.51Schwieters, Kuszewski, Tjandra and Clorestructure calculation
RefinementMethod: simulated annealing / Software ordinal: 7
Details: Docking was carried out between the two proteins using rigid body simulated annealing with a flexible linker between diubiquitin domains. Initial structures were 3H7P and 2MJB for ubiquitin, ...Details: Docking was carried out between the two proteins using rigid body simulated annealing with a flexible linker between diubiquitin domains. Initial structures were 3H7P and 2MJB for ubiquitin, and 1S1Q for Tsg101 UEV. Distal domain and Tsg101 backbone were fixed to 1S1Q position. Following initial docking, the lowest energy structure in each of the two clusters was used as input for refinement, with an ensemble number of 2.
NMR representativeSelection criteria: target function
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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